CN109536574A - A kind of colorimetric method of simple detection fibrin ferment - Google Patents
A kind of colorimetric method of simple detection fibrin ferment Download PDFInfo
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- CN109536574A CN109536574A CN201811249045.7A CN201811249045A CN109536574A CN 109536574 A CN109536574 A CN 109536574A CN 201811249045 A CN201811249045 A CN 201811249045A CN 109536574 A CN109536574 A CN 109536574A
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- 102000009123 Fibrin Human genes 0.000 title claims abstract description 74
- 108010073385 Fibrin Proteins 0.000 title claims abstract description 74
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 title claims abstract description 74
- 229950003499 fibrin Drugs 0.000 title claims abstract description 74
- 238000001514 detection method Methods 0.000 title abstract description 30
- 238000004737 colorimetric analysis Methods 0.000 title abstract description 14
- 239000013302 MIL-88A Substances 0.000 claims abstract description 53
- 108090000190 Thrombin Proteins 0.000 claims abstract description 31
- 229960004072 thrombin Drugs 0.000 claims abstract description 31
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 17
- GOMLCFUVZKLQCO-HTLAMOOLSA-N thrombin aptamer Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@@H]2[C@H](O[C@H](C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2COP(O)(=O)O[C@H]2[C@H]([C@@H](O[C@@H]2CO)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)N2C3=C(C(NC(N)=N3)=O)N=C2)O)[C@@H](OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=C(C(NC(N)=N3)=O)N=C2)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=C(C(NC(N)=N3)=O)N=C2)O)C1 GOMLCFUVZKLQCO-HTLAMOOLSA-N 0.000 claims abstract description 13
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- 229960005348 antithrombin iii Drugs 0.000 description 2
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- NQXWGWZJXJUMQB-UHFFFAOYSA-K iron trichloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].Cl[Fe+]Cl NQXWGWZJXJUMQB-UHFFFAOYSA-K 0.000 description 2
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- 108010094028 Prothrombin Proteins 0.000 description 1
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- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
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- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- HTNUJXYXLFKFKU-UHFFFAOYSA-N gold hydrogen peroxide Chemical compound [Au].OO HTNUJXYXLFKFKU-UHFFFAOYSA-N 0.000 description 1
- -1 gold ion Chemical class 0.000 description 1
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- 229910052742 iron Inorganic materials 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
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- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Spectroscopy & Molecular Physics (AREA)
- General Health & Medical Sciences (AREA)
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- Wood Science & Technology (AREA)
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- General Physics & Mathematics (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of colorimetric method of simple detection fibrin ferment, there is the characteristic of Mimetic Peroxidase in acid condition using metal organic framework compound Fe-MIL-88A, the fibrin ferment tetramer is generated by Thrombin aptamer and blood coagulation enzyme reaction, then again with Fe-MIL-88A and 3,3 ', 5,5 '-tetramethyl benzidine (TMB)-H2O2System carries out catalytic oxidation, using ultraviolet spectrophotometry and colorimetric method for determining concentration of thrombin.The present invention acts on the specific binding of substrate using the mimetic enzyme catalysis performance and aptamer of Fe-MIL-88A, can sensitive, accurate, specific and visual detection fibrin ferment.
Description
Technical field
The present invention relates to technical field of analysis and detection, more particularly, to a kind of colorimetric method of simple detection fibrin ferment, specifically
For the colorimetric method that metal organic framework compound material Fe-MIL-88A is used to detect fibrin ferment.
Background technique
Fibrin ferment (thrombin) is a kind of very important serine protease in body blood coagulation system, it is by 308
Amino acid residue composition, molecular weight 37kD.Fibrin ferment has in blood coagulation system promotees solidifying and anticoagulant dual function, in inflammation tune
Section, angiogenesis play an important role in tissue repair and oncobiology, also have in central nervous system pathology and
Physiological action.Fibrin ferment is considered as the important measurement index and some diseases diagnosis (such as Lung metastases and cartilage of clotting mechanism
Synovial membrane inflammation) biological marker.Therefore, fibrin ferment detects the monitoring to the early diagnosis of clinical disease, disease, curative effect
With assessment etc. all have very important significance.
The method of detection fibrin ferment mainly has red, orange, green, blue, yellow (ROGBY), fluorescence method immunoassay and aptamer method at present.By
In aptamer sensor due to having many advantages, such as that combining target species are wide, affinity is high, high specificity, at low cost,
It is widely used in analysis detection field.Wherein, colorimetric method is to be measured to determine by comparing measurement coloring matter solution colour depth
The method of constituent content, may be implemented Visual retrieval, show greatly advantage in fibrin ferment detection application aspect.
Patent CN201510073478.1 provides a kind of based on gold-hydrogen peroxide variable color system and rolling circle amplification phase
In conjunction with blood coagulation enzyme assay method.In the presence of hydrogen peroxide, gold ion is reduced into nanogold, solution presents red；When solidifying
Hemase aptamer and annular template interconnect, and carry out rolling circle amplification and generate DNA enzymatic.Since DNA enzymatic can be catalyzed peroxidating
Hydrogen decomposes, and reduces concentration of hydrogen peroxide, forms the nanogold of reunion, and blue is presented in solution.The rolling circle amplification that the invention uses
Technology, complex steps, reaction time are long.
Patent CN201180059028.7 provides a kind of for comprising Antithrombin III (AT-III) and fibrin ferment
The method that in vitroimmunoassay is carried out to fibrin ferment in sample.First by the substrate binding site of fibrin ferment and identification fibrin ferment
Small molecule contact；Secondly, fibrin ferment is contacted with Thrombin specificity antibody.Horizontal by measurement binding antibody realizes blood coagulation
The detection of enzyme.This kind of method, cumbersome, required biological reagent is more, and condition of storage is harsh.
Metal organic framework compound (Metal-Organic Framework, MOF) is one kind developed in recent years
The polymer with infinite network structure connected by inorganic metal ion and organic ligand by unlimited coordination mode is more
Porous materials.Since metal organic framework compound has many advantages, such as porosity, hole controllability, is easy to functionalization, in molecule
The fields such as detection show good application prospect (Li Song, the design synthesis and visualization inspection of metal-organic framework crystalline material
Survey metal ion probe research [D], South China Science & Engineering University, 2015).
Studies have shown that part MOF material has Mimetic enzyme of peroxidase catalytic performance.Studies have found that Fe-MIL-53
With Mimetic Peroxidase catalytic performance, and be used for hydrogen peroxide detection, have good detection effect (Ai L, Li L,
Zhang C, Fu J, Jiang J.MIL-53 (Fe): a metal-organic framework with intrinsic
peroxidase-like catalytic activity for colorimetric biosensing[J]
.Chemistry.2013,19 (45): 15105-8.).In addition scholar has studied Fe-MIL-88NH2Peroxidase work
Property and be applied to glucose detection (Liu YL, Zhao XJ, Yang XX, Li YF.A nanosized metal-organic
framework of Fe-MIL-88NH(2)as a novel peroxidase mimic used for colorimetric
Detection of glucose [J] .The Analyst.2013,138 (16): 4526-31.).
Aptamer is that the energy specificity screened in vitro by index concentration Fas lignand system evolution technology is known
A kind of single-stranded oligonucleotide of other target molecule.Aptamer is with specific bond object type is wide, affinity is high, specific
By force, the advantages that easily modifying, is widely used in analysis detection field (Zheng Jing, He Pingang, the aptamer biosensor of square Yu
[J] chemical progress, 2009,04:732-738.).But nucleotide aptamer is mainly used for detecting biological micromolecule at present, thinks
The difficult of detection also many technical aspects for carrying out large biological molecule needs to overcome.
Summary of the invention
In view of the above-mentioned problems existing in the prior art, this application provides a kind of colorimetric methods of simple detection fibrin ferment.This
Invention uses sensitive Colorimetric System, and the blue solution generated using Fe-MIL-88A catalyzing hydrogen peroxide oxidation TMB is directly sentenced
The presence of disconnected fibrin ferment；It is simple and easy it is not necessary that aptamer is marked in advance, it is not necessarily to complex instrument equipment.
Technical scheme is as follows:
A method of detection fibrin ferment, which comprises the steps of:
(1) sample to be detected and Thrombin aptamer are mixed to get the fibrin ferment tetramer；
(2) the fibrin ferment tetramer is mixed with Fe-MIL-88A solution；
(3) 3,3 ', 5 are added in the mixed liquor obtained to step (2), 5 '-tetramethyl benzidines, that is, TMB, H2O2Solution and vinegar
Sour sodium buffer, carries out catalysis reaction；
(4) sulfuric acid solution is added, absorbance is surveyed using ultraviolet specrophotometer, by test result and standard fibrin ferment curve
Comparison, obtains the concentration of thrombin of sample to be tested.
Wherein, the concrete operation method of step (1) are as follows:
The 5 '-TCT CTC AGT CCG TGG TAG GGC AGG of Thrombin aptamer for being 1 μM by 100 μ L concentration
TTG GGG TGA CT-3 ' reacts 1 hour with 50 μ L samples to be detected in D-PBS buffer at 25 DEG C, obtains blood coagulation
The enzyme tetramer；
The D-PBS pH of buffer is 7.4, and group becomes potassium chloride 2.67mmol/L；Dipotassium hydrogen phosphate 8.1mmol/L；Phosphorus
Acid dihydride sodium 1.47mmol/L；Sodium chloride 138mmol/L；Magnesium chloride 5mmol/L.
The concrete operation method of step (2) are as follows:
The Fe-MIL-88A solution that 10 μ L concentration are 0.4mg/mL, concussion reaction 3 are added in the fibrin ferment tetramer
Minute.
The concrete operation method of step (3) are as follows:
The H that TMB, 10 μ L concentration that 100 μ L concentration are 10mM are 10mM is added in the mixed liquor obtained to step (2)2O2It is molten
The HAc-NaAc buffer of the pH 3.0 of liquid and 730 μ L react 20 minutes at 40 DEG C.
The concrete operation method of step (4) are as follows:
The H that 50 μ L concentration are 2mM is added into mixed liquor after reaction2SO4Solution reads absorbance at 451nm
Value.
Step (4) the standard fibrin ferment curve, which refers to, carries out step (1)~(3) for the thrombin solution of normal concentration
After processing, sulfuric acid solution is added, absorbance is surveyed using ultraviolet specrophotometer, using corresponding concentration of thrombin as abscissa, with this
Concentration thrombin solution absorbance at ultra-violet absorption spectrum 451nm is the curve that ordinate is drawn.Standard curve is specifically drawn
Method processed are as follows:
(1) synthesis of Fe-MIL-88A: Iron(III) chloride hexahydrate and fumaric acid are dissolved in ultrapure water first, then
Entire homogeneous phase solution, which is transferred in reaction kettle, heats reaction.After being cooled to room temperature, reactant water and ethyl alcohol clean repeatedly, centrifugation
Product Fe-MIL-88A powder is obtained after being dried overnight in a vacuum drying oven afterwards.It weighs the powder and is dissolved in ultrasound in ultrapure water
Fe-MIL-88A solution is formed after mixing.
The ultrapure water refers to that the conductivity prepared with Milli-Q ultrapure water instrument is 18.2M Ω cm-1Pure water.
The concentration of the Fe-MIL-88A solution is 0.4mg/mL；Storage temperature: normal temperature storage.
(2) formation of the fibrin ferment tetramer: Thrombin aptamer and fibrin ferment are reacted in D-PBS solution, are formed
Fibrin ferment tetramer solution, includes the following steps:
I, Thrombin aptamer freeze-dried powder is centrifuged 10 minutes under the conditions of 10000rmp, D-PBS buffer solution is added,
It is configured to Thrombin aptamer titer.
The concentration of the Thrombin aptamer titer is 1 μM, condition of storage are as follows: storage temperature: 0-4 DEG C；When storage
Between: within 1 month.
The D-PBS is achieved through the following technical solutions:
II, thrombin standard substance solution is weighed, with ultrapure water dissolution and constant volume, preparing standard solution.
The concentration of the thrombin standard solution is 2 μm of ol/L.
The condition of storage of the thrombin standard solution are as follows: storage temperature: 0-4 DEG C；Storage time: within 1 month.
III, the thrombin standard solution pipetted in the step II of different volumes respectively are divided with ultrapure water dilution and constant volume
It is not formulated as a series of thrombin standard solution of various concentrations.
The standard solution should cover the concentration range of fibrin ferment in sample to be tested, can be i.e. with i.e. use.
The thrombin standard solution of Thrombin aptamer and various concentration is mixed, is reacted 1 hour at room temperature, is formed
Fibrin ferment tetramer standard solution.
(3) TMB catalysis reaction: the fibrin ferment four for respectively generating Fe-MIL-88A solution in step (1), step (2)
Aggressiveness standard solution carries out mixing and carries out catalysis reaction.
I, TMB standard substance is weighed, with ethanol solution dissolution and constant volume, configures TMB solution.
The concentration of the TMB solution is 10mmol/L.
The condition of storage of the TMB solution are as follows: storage temperature: 0-4 DEG C；Storage time: within 1 month.
II, H is pipetted2O2Solution, with ultrapure water dilution and constant volume, preparing standard solution.
The H2O2The concentration of standard solution is 10mmol/L.
The H2O2The condition of storage of standard solution are as follows: normal temperature storage.
III, acetic acid and acetic acid sodium crystal are weighed, with ultra-pure water solution and constant volume, configures sodium acetate buffer (pH=
3)。
IV, after being added after mixing Fe-MIL-88A and fibrin ferment tetramer standard solution, TMB, H are sequentially added2O2, vinegar
Sour sodium buffer, 40 DEG C are reacted 20 minutes.
(4) detect: after reaction, into reaction solution be added sulfuric acid terminate liquid, after UV-1700 UV absorption be divided
Photometric determination absorption spectrum.
The concentration of the sulfuric acid terminate liquid is 2mol/L.
(5) standard curve is drawn.
Corresponding standard working curve is drawn according to the ultra-violet absorption spectrum that step (4) obtains, calculates separately to obtain blood coagulation
The regression equation of enzyme standard working curve.
In the standard curve, using at ultra-violet absorption spectrum 451nm absorbance as ordinate (Y-axis), corresponding blood coagulation
Enzyme concentration is abscissa (X-axis).
The detection of ultraviolet specrophotometer carries out at room temperature.Spectral scan is opened from wave-length coverage from 400nm
Begin to 800nm to terminate.
After obtaining above-mentioned standard curve, by the processing result and standard curve control of sample to be tested, fibrin ferment can be obtained
Concentration.It can also be scanned without ultraviolet light spectrophotometer, but after terminating reaction, only compare by visually to test sample
The color of product and thrombin standard sample judges the concentration of sample to be tested fibrin ferment by colorimetric method.
Metal organic framework compound material Fe-MIL-88A has Mimetic Peroxidase catalytic, can be catalyzed H2O2
TMB is aoxidized, solution becomes blue；After the completion of reaction, sulfuric acid terminate liquid is added, solution turns yellow.
The purpose of the present invention is to provide a kind of colorimetric methods of simple detection fibrin ferment, for accurate and reliable, operation to be simple
Realize the detection of colorimetric method fibrin ferment in ground.To achieve the above object, the present invention is raw using Thrombin aptamer and blood coagulation enzyme reaction
At the fibrin ferment tetramer, then with a kind of iron content organic framework compounds Fe-MIL-88A and TMB-H2O2Catalysis reaction is carried out, is used
The concentration of ultraviolet spectrophotometer method measurement fibrin ferment.
By sample to be tested formed the fibrin ferment tetramer and with Fe-MIL-88A, TMB, H2O2After equal solution reactions, using purple
The result of outer spectrophotometer detection need to obtain the concentration of thrombin number of sample to be tested with the data comparison of thrombin standard curve
According to.
The present invention is beneficial to be had the technical effect that
The advantages of present invention combination metal organic framework compound mimetic enzyme catalysis performance and aptamer it is highly selective
And specificity, establish a kind of visualization blood coagulation enzyme assay method simple, sensitive, specificity is high.The present invention is loaded using MOF
Fibrin ferment aptamer forms void effect, realizes fibrin ferment detection, constructs point specifically by MOF and aptamer
The duct of sub switch.The duct of molecular switch is opened through aptamer in the presence of no target, and small molecule TMB can be with
Into duct, acts on and developing the color by MOF mimetic enzyme catalysis；In the presence of having target molecule, aptamer identifies molecule, forms four
Aggressiveness, duct are closed, and small molecule TMB cannot enter duct, MOF enzymatic miopragia or disappearance, cannot be developed the color, thus real
Now detect.How under suitable conditions technological difficulties therein are rapid build ducts, and it is tired that this is that the present invention overcomes technologies
Difficult place.
The range of linearity that the present invention measures fibrin ferment is 20-80nmol/L, and detection is limited to 0.8nmol/L.Utilize different lifes
The amino acid of object macromolecular and different isoelectric points shows selection of the MOF- aptamer sensor to fibrin ferment as interfering substance
Property is good；Using fibrin ferment recovery testu in serum, the rate of recovery of fibrin ferment is measured between 99.3% to 100.4%,
Relative standard deviation is between 0.3% to 1%.
Detailed description of the invention
Fig. 1 is the transmission electron microscope picture of Fe-MIL-88A；
Fig. 2 is the XRD spectrum of Fe-MIL-88A；
Fig. 3 is the ultraviolet spectra response diagram and calibration curve of fibrin ferment in detection method；
Fig. 4 is the changing rule of the thrombin solution color of various concentration；
Fig. 5 is the anti-interference schematic diagram of MOF- aptamer sensor；
Fig. 6 is the mimetic enzyme catalysis performance test result of Fe-MIL-88A described in comparative example 1: a) TMB；b)TMB+H2O2；c)
TMB+Fe-MIL-88A；d)Fe-MIL-88A+H2O2+TMB；Interior illustration: the color change comparison diagram of solution, vial a are corresponding
It is line a, it is other similar；
Fig. 7 is the condition optimizing result of Fe-MIL-88A mimetic enzyme catalysis described in comparative example 2: A) H2O2Concentration；B)Fe-
MIL-88A concentration；C) pH and D) temperature.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with the drawings and specific embodiments
The invention will be further described.It should be appreciated that preferred embodiments described herein is merely to illustrate and explain the present invention, and
It is not used in the restriction present invention.
Metal organic framework compound Fe-MIL-88A, Thrombin aptamer and fibrin ferment is cleverly utilized in the present invention
Interaction relationship between each substance designs a kind of colorimetric method that can be used in detecting thrombin amount.Fe-MIL-88A can
In a certain amount of H2O2TMB colour developing is catalyzed under the conditions of existing, solution becomes blue from colourless.Under certain conditions, fibrin ferment core
The capture prothrombin molecule of the sour aptamer property of can choose forms the fibrin ferment tetramer.The fibrin ferment tetramer is added to Fe-MIL-
88A and TMB-H2O2In system, under the action of electrostatic attraction, the fibrin ferment tetramer can be wrapped in around Fe-MIL-88A, be hindered
Electron transmission between Fe-MIL-88A and TMB inhibits the progress of oxidation reaction, it is non-discolouring to show as solution to be measured.Pass through light splitting
The highly sensitive specific detection to fibrin ferment may be implemented in photometry and colorimetric method.
Embodiment 1
(1) synthesis of Fe-MIL-88A
First by 5mM Iron(III) chloride hexahydrate (FeCl3·6H2O it) is dissolved in 25mL ultrapure water with 5mM fumaric acid, then
Entire homogeneous phase solution, which is transferred in 60mL reaction kettle, is heated to 65 DEG C of reactions 12 hours.After being cooled to room temperature, reactant water and
Ethyl alcohol cleans repeatedly, obtains product Fe-MIL-88A powder after being dried overnight in vacuum oven (being lower than 80 DEG C) after centrifugation
End.It weighs the 4.0mg powder and is dissolved in 10.0mL ultrapure water formation 0.4mg/mL Fe-MIL-88A solution after ultrasonic mixing.
It will be seen from figure 1 that Fe-MIL-88A is a kind of fusiform crystal.Figure it is seen that Fe-MIL-88A has
There is specific crystal structure.
(2) prepared by fibrin ferment MOF- aptamer mixture
100 μ L Thrombin aptamer, 5 '-TCT CTC AGT CCG TGG TAG GGC AGG TTG GGG TGA CT-
3 ' (concentration is 1 μM) and the fibrin ferment of 50 μ L various concentrations react 1 hour at room temperature.After reaction, to mixed liquor
10 μ LFe-MIL-88A (0.4mg/mL) of middle addition, 730 μ LHAc-NaAc buffers (pH 3.0), 100 μ L TMB solution
(10mM, ethyl alcohol are solvent) and 10 μ L H2O2(10mM), 40 DEG C are reacted 20 minutes.50 μ L are added into mixed liquor after reaction
H2SO4Absorption spectrum is measured in UV--1700 Ultravioblet spectrophotometer after (2M).
(3) Specification Curve of Increasing
When drawing standard curve, with ultrapure water dilution and constant volume concentration is the thrombin solution of 2 μm of ol/L, is formulated as respectively
A series of thrombin standard solution of various concentrations.
The detection of ultraviolet specrophotometer carries out at room temperature.Spectral scan wavelength since 400nm to
800nm terminates.Fe-MIL-88A aoxidizes TMB reaction, and solution is made to become blue.The judgment basis of colorimetric method, i.e., according to solution indigo plant
The depth of color judges the concentration range of fibrin ferment, and color is deeper, and concentration of thrombin is smaller.But blue solution is in ultraviolet light
There is higher background value in spectrum, therefore, in UV spectrophotometer measuring method, after reaction, is added to blue solution
H28O4(2M) is used as reaction terminating liquid.With the addition of sulfuric acid solution, solution is turned yellow from blue at once, is gone out at 451nm
Existing characteristic absorption peak.
Using absorbance at ultra-violet absorption spectrum 451nm as ordinate (Y-axis), the concentration of corresponding thrombin standard solution is
Abscissa (X-axis), draws corresponding standard working curve, and the regression equation of thrombin standard working curve is calculated.Fig. 3 is
The ultraviolet spectra response diagram and calibration curve of fibrin ferment.
Under the influence of the fibrin ferment tetramer, Fe-MIL-88A is catalyzed H2O2Aoxidize TMB colour developing reaction and fibrin ferment it is dense
Spend it is in a linear relationship, can establish accordingly visualization fibrin ferment determination method.Under optimum reaction condition, oxidation
The concentration of absorbance and fibrin ferment of the TMB at 451nm has preferable linear relationship in a certain range.Its linear detection range
For 10nM-80nM (R2=0.9906), detection is limited to 0.8nM, and (LOD=3SD/S, wherein SD is the deviation of blank sample, and S is mark
The numerical value of the slope of directrix curve, each point in standard curve is the average value of 3 measurement results).
Fig. 4 is color camera corresponding to the fibrin ferment of system of determination various concentration concentration.With the increasing of concentration of thrombin
Greatly, the blue of solution is more and more shallow.
(4) colorimetric determination
Sample to be tested is handled according to above-mentioned steps (1), the method for (2), then uses Ultravioblet spectrophotometer
Absorbance data is detected, is compared with the thrombin standard solution colour (i.e. Fig. 4) after standard curve (i.e. Fig. 3) or reason,
Obtain the concentration of thrombin of sample to be tested.
Embodiment 2: anti-interference test
To probe into the method for the present invention to the anti-interference ability of the amino acid of other potential interference protein and different isoelectric points,
We are to bovine serum albumin (BSA), immunoglobulin G (IgG), lysozyme (lysozyme), lysine (lysine), arginine
(arginine), glycine (glycine), sodium glutamate (L-sodium glutamate, LSG) have carried out parallel disturbed test
Experiment.From fig. 5, it can be seen that only existing for the fibrin ferment under the conditions of, absorbance reduced rate significantly increases, and solution almost becomes
It is colourless.Under the conditions of existing for other high molecular weight proteins or the amino acid of various isoelectric points, the analogue enztme of Fe-MIL-88A is urged
The property changed does not receive apparent inhibiting effect, and solution is still blue.The experimental results showed that detection method of the invention is with good
Good interference free performance and fibrin ferment evident characteristics.
Embodiment 3: mark-on is tested in serum
It uses and the fibrin ferment of standard is added in Standard entertion normal direction cow's serum to be configured to the blood serum sample of various concentration.Blood
It is to remove left next blood plasma after fibrin after blood clotting clearly.The chemical component of serum is similar with blood plasma, but does not contain
Blood coagulating protein.Each group of mark-on experiment all repeats 3 groups of parallel laboratory tests, and the final data in table 1 is being averaged for 3 groups of parallel laboratory tests
Value.The result shows that the rate of recovery of fibrin ferment is between 99.3% to 100.4%, relative standard deviation between 0.3% to 1%,
Accuracy and repeatability of the present invention are preferable, can satisfy quantitative needs.
1 serum mark-on of table tests the thrombin samples rate of recovery
Comparative example 1:
By being catalyzed H2O2Oxidation TMB reacts to verify the mimetic enzyme catalysis performance of Fe-MIL-88A.Reaction condition: TMB,
1mM；H2O2, 100 μM；Fe-MIL-88A, 4 μ g/mL；HAc-NaAc(pH 3.0)；40 DEG C are reacted 20 minutes.
As a result as shown in Fig. 6 line a and line b, in only H2O2Suction under conditions of addition, at 652nm ultra-violet absorption spectrum
Peak is received almost without what variation, TMB solution is still that colourless (Fig. 6 bottle b) shows TMB almost without aoxidizing.Once Fe-
MIL-88A is added to TMB-H2O2In system, the absorption peak at 652nm ultra-violet absorption spectrum significantly increases (Fig. 6 line d), solution
(Fig. 6 bottle d) shows that Fe-MIL-88A can be very good oxidation matrix TMB to the apparent blue that becomes.
Comparative example 2:
Same horseradish peroxidase (HRP) is similar, the mimetic enzyme catalysis activity of Fe-MIL88A be heated temperature, pH value,
H2O2The influence of concentration and catalytic materials concentration.Therefore, this experiment has studied temperature, pH value, H respectively2O2Concentration and Fe-MIL-
Influence of the 88A concentration to Fe-MIL-88A mimetic enzyme catalysis.
Firstly, this experimental study Fe-MIL-88A is in different H2O2The catalytic activity of (100 μM of -400mM) under concentration.It will
The H of various concentration2O2Solution is added to containing 0.2mM TMB, 0.04mg/mL Fe-MIL-88A, sodium-acetate buffer (pH
3.0) it in mixed liquor, is reacted 20 minutes in 40 DEG C of water-baths.The H of high concentration as can be seen from Figure 7A2O2Can have one to catalysis
Fixed inhibiting effect, but the H of Fe-MIL-88A2O2Tolerable concentration is higher than HRP, shows the H in high concentration2O2Under the conditions of,
Fe-MIL-88A ratio HRP is more stable.
Then, influence of the concentration of this experimental study Fe-MIL-88A to catalysis reaction.The Fe- of 0.01-0.05mg/mL
MIL-88A is added to containing 0.2mM TMB, 400 μM of H2O2, in the mixed liquor of sodium-acetate buffer (pH 3.0), in 40 DEG C of water
20min is reacted in bath.When Fe-MIL-88A amount is 0.04mg/mL, catalytic activity reaches maximum (Fig. 7 B).
Then, influence of this experimental study pH value to catalysis reaction.0.2mM TMB, 400 μM of H2O2, 0.04mg/mL
Fe-MIL-88A is added in the sodium-acetate buffer of different pH value (2.5-6), is reacted 20 minutes in 40 DEG C of water-baths.Experiment
Show that Fe-MIL-88A can guarantee good catalytic effect (Fig. 7 C) in the range of pH value is 2.5-4.
Finally, 0.2mM TMB, 400 μM, 0.04mg/mL Fe-MIL-88A and sodium-acetate buffer (pH 3.0) are in difference
It is heated 20 minutes in the water-bath of temperature, to detect influence (Fig. 7 D) of the temperature to catalysis reaction.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (6)
1. a kind of method for detecting fibrin ferment, which comprises the steps of:
(1) sample to be detected and Thrombin aptamer are mixed to get the fibrin ferment tetramer；
(2) the fibrin ferment tetramer is mixed with Fe-MIL-88A solution；
(3) 3,3 ', 5 are added in the mixed liquor obtained to step (2), 5 '-tetramethyl benzidines, that is, TMB, H2O2Solution and sodium acetate
Buffer carries out catalysis reaction；
(4) sulfuric acid solution is added, absorbance is surveyed using ultraviolet specrophotometer, by test result and standard fibrin ferment curve pair
Than obtaining the concentration of thrombin of sample to be tested.
2. the method according to claim 1, wherein the concrete operation method of step (1) are as follows:
The 5 '-TCT CTC AGT CCG TGG TAG GGC AGG TTG of Thrombin aptamer for being 1 μM by 100 μ L concentration
GGG TGA CT-3 ' reacts 1 hour with 50 μ L samples to be detected in D-PBS buffer at 25 DEG C, obtains fibrin ferment four
Aggressiveness；
The D-PBS pH of buffer is 7.4, and group becomes potassium chloride 2.67mmol/L；Dipotassium hydrogen phosphate 8.1mmol/L；Di(2-ethylhexyl)phosphate
Hydrogen sodium 1.47mmol/L；Sodium chloride 138mmol/L；Magnesium chloride 5mmol/L.
3. the method according to claim 1, wherein the concrete operation method of step (2) are as follows:
Be added in the fibrin ferment tetramer 10 μ L concentration be 0.4mg/mL Fe-MIL-88A solution, concussion reaction 3 minutes.
4. the method according to claim 1, wherein the concrete operation method of step (3) are as follows:
The H that TMB, 10 μ L concentration that 100 μ L concentration are 10mM are 10mM is added in the mixed liquor obtained to step (2)2O2Solution with
And 730 μ L pH 3.0 HAc-NaAc buffer, react 20 minutes at 40 DEG C.
5. the method according to claim 1, wherein the concrete operation method of step (4) are as follows:
The H that 50 μ L concentration are 2mM is added into mixed liquor after reaction2SO4Solution reads absorbance value at 451nm.
6. the method according to claim 1, wherein step (4) the standard fibrin ferment curve refers to standard
After the thrombin solution of concentration carries out the processing of step (1)~(3), sulfuric acid solution is added, is surveyed and is inhaled using ultraviolet specrophotometer
Luminosity, using corresponding concentration of thrombin as abscissa, with the concentration thrombin solution, absorbance is at ultra-violet absorption spectrum 451nm
The curve that ordinate is drawn.
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| CN110095420A (en) * | 2019-05-08 | 2019-08-06 | 国家纳米科学中心 | A kind of detection method and its application of concentration of hydrogen peroxide |
| CN110567953A (en) * | 2019-10-12 | 2019-12-13 | 山西师范大学 | Used for detecting Fe in environmental water sample and serum2+Content visual detection kit and detection method thereof |
| CN111504986A (en) * | 2020-04-03 | 2020-08-07 | 上海理工大学 | Method for rapidly detecting diamine biogenic amine |
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Cited By (4)
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| CN110095420A (en) * | 2019-05-08 | 2019-08-06 | 国家纳米科学中心 | A kind of detection method and its application of concentration of hydrogen peroxide |
| CN110567953A (en) * | 2019-10-12 | 2019-12-13 | 山西师范大学 | Used for detecting Fe in environmental water sample and serum2+Content visual detection kit and detection method thereof |
| CN110567953B (en) * | 2019-10-12 | 2022-07-01 | 山西师范大学 | Used for detecting Fe in environmental water sample and serum2+Content visual detection kit and detection method thereof |
| CN111504986A (en) * | 2020-04-03 | 2020-08-07 | 上海理工大学 | Method for rapidly detecting diamine biogenic amine |
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